Central and peripheral type benzodiazepine ligands displace [3H][3-ME-HIS2]TRH from its binding sites in the brain and the anterior pituitary and antagonize the effect of TRH in the rat duodenum.

The effects of central (clonazepam, an agonist, and FG 7142, an inverse agonist), mixed (diazepam) or peripheral type (Ro 5-4864) benzodiazepine receptor ligands on the action of TRH on the transmurally stimulated rat duodenum and binding of [3H][3-Me-His2] TRH in the rat anterior pituitary, hypothalamus, cortex and brainstem have been studied. TRH dose-dependently inhibited the contractions of transmurally stimulated rate duodenum. Clonazepam (5 x 10(-6) M), diazepam (10(-5) M), Ro 5-4864 (10(-5) M) or FG 7142 (10(-5) M) attenuated the response of TRH in the rat duodenum. The action of these compounds was antagonized neither by the central type benzodiazepine antagonist flumazenil nor by peripheral type antagonist PK 11195 but instead PK 11195 itself counteracted TRH. TRH displaced [3H][3-Me-His2]TRH with Ki-values ranging 0.08 to 0.31 microM. Ki-values for clonazepam diazepam, Ro 5-4864, PK 11195 and FG 7142 ranged 6-117 microM, 3-23 microM, 20-67 microM, 20-40 microM and 260-420 microM, respectively, demonstrating fairly weak affinity to TRH-receptors. In saturation experiments, clonazepam and PK 11195 significantly increased KD but not Bmax of the labelled ligand while Ro 5-4864 increased both KD and Bmax. This indicates that all these compounds competitively inhibit the binding of [3H][3-Me-His2]TRH in the CNS which may also be the mechanism for their antagonism of the effect of TRH in the rat duodenum.     

Chronic ethanol treatment alters the behavioral effects of Ro 15-4513, a partially negative ligand for benzodiazepine binding sites

Pentylenetetrazol (PTZ)-induced convulsion were studied in control, chronic ethanol-maintained, and ethanol-withdrawal rats. The convulsive doses of PTZ varied among the different groups of rats. Ethanol-maintained rats required higher doses of PTZ to produce convulsions, compared to control and ethanol-withdrawal rats. The partially negative ligands for benzodiazepine binding sites, Ro 15-4513 (2 mg/kg, i.p.) and FG 7142 (20 mg/kg, i.p.) produced proconvulsant effect in saline (control) and ethanol-withdrawal rats as they potentiated the effect of subconvulsive dose of PTZ. A higher dose of Ro 15-4513 (4 mg/kg, i.p.), but not FG 7142 (up to 80 mg/kg, i.p.), also produced proconvulsant effect in ethanol-maintained rats. Furthermore, Ro 15-4513 (5, 10 mg/kg, i.p.), but not FG 7142 (up to 80 mg/kg, i.p.), produced clonic-tonic seizures of short duration in ethanol-withdrawal rats. These effects of Ro 15-4513 and FG 7142 were reversed by diazepam (2 mg/kg, i.p.), as well as by the GABA-neutral Ro 15-1788 (10 mg/kg, i.p.), thereby, indicating the involvement of central benzodiazepine receptors in the action of Ro 15-4513 and FG 7142. These observations suggest that chronic ethanol treatment selectively alters the receptor sensitivity to Ro 15-4513, an ethanol antagonist and partially negative ligand for BZ sites, and this observation supports the notion that ethanol effects are more susceptible to reversal by the imidazobenzodiazepine as compared to other negative ligand for BZ binding sites.     

Immunomodulatory effect of peripheral benzodiazepine receptor ligands on human mononuclear cells.

Immunomodulatory effect of ligands active at the peripheral benzodiazepine receptor (PBR) was examined in human peripheral blood mononuclear cells (PBMC). Ro5-4864, PK11195 and diazepam suppressed phytohemagglutinin (PHA) and concanavalin A (ConA) induced proliferation of PBMC. All three ligands inhibited interleukin-3-like activity (IL-3-LA) secretion, while the production of interleukin-2 (IL-2) was inhibited by Ro5-4864 and diazepam only. The selective central benzodiazepine ligand clonazepam did not affect the cellular immune functions examined. Our results indicate an in-vitro immuno-suppressive activity of peripheral and mixed, but not central type benzodiazepine ligands.     

Suppression of natural killer cell activity by FG 7142, a benzodiazepine receptor "inverse agonist"

A dose-dependent (5-50 mg/kg) suppression of natural killer (NK) cell activity was observed 2 h after administration of the benzodiazepine receptor "inverse agonist" FG 7142 (N-methyl-beta-carboline-3-carboxamide), and was still manifest 24 h later. Addition of FG 7142 (1-1000 nM) to the 4 h 51 Cr release assay did not affect NK cell activity. Pretreatment of mice with the benzodiazepine receptor antagonist Ro 15-1788 (10 mg/kg) blocked FG 7142-induced suppression of NK cell activity, but had no effect when administered alone. The suppression of NK cell activity by FG 7142, a compound which produces a syndrome resembling stress or anxiety in both animals and man, provides further evidence that the central nervous system pathways subserved by the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex") may play a role in the modulation of immune function.     

The anorectic effect of FG 7142, a partial inverse agonist at benzodiazepine recognition sites, is reversed by CGS 8216 and clonazepam but not by food deprivation

The benzodiazepine partial inverse agonist N′-methyl-β-carboline-3-carboxamide (FG 7142; 5.0 and 10.0 mg/kg, i.p.) produced a dose-dependent reduction in the consumption of a familiar, highly palatable diet by non-food-deprived male rats. At dose levels which exhibited no significant intrinsic effects, the benzodiazepine receptor antagonist 2-phenylpyrazolo-[4,3-c]-quinoline-3(5H)-one (CGS 8216; 1.25–5.0 mg/kg, i.p.) reversed the anorectic effect of FG 7142. When clonazepam and FG 7142 were given in combination, mutual cancelling of their opposite effects occurred. These results are consistent with an action of FG 7142 at benzodiazepine recognition sites to reduce the level of palatable food consumption, and imply that a bidirectional control of food intake via benzodiazepine recognition sites can be achieved. The anorectic effect of FG 7142 was not reversed by 24-h food deprivation, indicating a possible separation from the effects of hunger mechanisms.     

Inhibition of cold-induced TSH release by benzodiazepines

Diazepam (1–5 mg/kg, i.p.) did not alter basal thyroid-stimulating hormone (TSH) release in rats; however, both diazepam (2 mg/kg, i.p.) and clonazepam (1 mg/kg, i.p.) blocked the 3–5-fold increase in TSH induced by cold exposure. The benzodiazepine antagonist, Ro15-1788 (10 mg/kg, i.p.) was effective in reversing clonazepam inhibition of cold-induced TSH release, but when administered alone had no effect on either basal TSH levels or the rise in TSH following cold exposure. Diazepam did not affect the ability of TRH to increase TSH secretion.     

Chronic treatment with diazepam or the inverse benzodiazepine receptor agonist FG 7142 causes differential changes in the effects of GABA receptor stimulation

Daily treatment of mice with diazepam leads to the development of tolerance to the anxiolytic and anticonvulsant effect of the benzodiazepine, while daily treatment with the proconvulsant benzodiazepine receptor inverse agonist FG 7142 produces sensitization to its effects in that seizures develop (chemical kindling). In the present study, the effects of GABA receptor stimulation were studied 2 days after termination of 13 days treatment with diazepam, 20 mg/kg i.p./day, and FG 7142, 40 mg/kg i.p./day. For GABA receptor stimulation, the GABA agonist progabide was chosen because among several GABA receptor stimulants tested it was the only compound that induced increases in seizure threshold in non-toxic doses. Using the threshold for maximal (tonic extension) electroconvulsions as a measure for anticonvulsant efficacy, the anticonvulsant effect of progabide (100 mg/kg i.p.) was unchanged after chronic treatment with diazepam but was lost in FG 7142 kindled animals. Conversely, the hypothermic effect of progabide was reduced after treatment with diazepam but not with FG 7142. Baseline seizure threshold was unchanged 2 days after chronic administration of diazepam but increased in the FG 7142 pretreated mice. The data indicate that tolerance to benzodiazepines and kindling by FG 7142 are associated with different changes in GABA receptor function.     

'Peripheral' Benzodiazepine Receptor Ligands Stimulate Prolactin Release in the Rat

High concentrations of ‘peripheral’ benzodiazepine binding sites have been described in the pituitary gland and in several other endocrine glands, such as the adrenal glands and the testes. The role played by these receptors on the regulation of the endocrine system is largely unknown. In this study, we report the effects of two ligands of the ‘peripheral’ benzodiazepine receptor, Ro5–4864 and PK 11195, on prolactin (PRL) release in the adult male rat. Ro5-4864 stimulated PRL release with half maximal and maximal stimulatory doses of about 0.6 and 1.2 mg/kg, respectively. Pretreatment with the ‘peripheral’ benzodiazepine receptor antagonist PK 11195 did not have any effect on Ro5-4864-induced PRL release. Accordingly, PK 11195, given alone, stimulated PRL release in a dose-dependent fashion. To examine whether the stimulatory effect of Ro5-4864 and PK 11195 on PRL release was due to a direct effect of these compounds at the pituitary gland, we used primary cultures of anterior pituicytes. Neither Ro5-4864 nor PK 11195 had an effect on basal PRL release nor were these agents capable of modulating thyrotropin-releasing hormone-stimulated or dopamine-inhibited PRL release. These findings suggest that administration of agents that interact with the ‘peripheral’ benzodiazepine binding receptor cause PRL release without directly stimulating the pituitary gland. We speculate that Ro5–4864 and PK 11195 increase plasma PRL levels by modulating brain release of neurotransmitters and/or neuropeptides involved in the regulation of PRL release.     

Effect of benzodiazepines on the proliferation of mouse spleen lymphocytes in vitro

Summary The effect of several benzodiazepines (clonazepam, diazepam, Ro 5-4864, Ro 15-1788) and two pineal gland indoleamines (N-acetylserotonin, melatonin) on the spontaneous proliferation of mouse spleen lymphocytes was estimated in vitro by the 3 H-thymidine uptake assay. It was found that diazepam and Ro 5-4864 (a selective peripheral-type benzodiazepine receptor ligand) produced the concentration-dependent inhibition of 3 H-thymidine incorporation into the DNA of these cells. Ro 15-1788, a specific central-type receptor ligand, evoked a slight inhibitory effect in a high concentration (10^–4M), whereas clonazepam did not produce any significant inhibition. When Ro 5-4864 was tested in combination with diazepam, the inhibition of lymphocyte proliferation did not exceed the effect of diazepam given alone. Ro 15-1788 was unable to reverse the inhibitory action of diazepam in the same experimental conditions. Melatonin and its precursor N-actetylserotonin tested in the concentration range of 10^–4–10^–8 M had no significant influence on the spleen lymphocyte DNA replication in our assay system.These data suggest that diazepam inhibition of lymphocyte proliferation is mediated by peripheral-type sites. Additionally, the fact that melatonin and N-acetylserotonin were unable to affect 3 h-thymidine incorporation argues against any benzodiazepine receptor mediated effect of pineal indoleamines on a cellular proliferation.     

Enhanced sensitivity to β-carboline inverse agonists in rats chronically treated with FG 7142

The biochemical and behavioural effects of the chronic administration of the beta-carboline inverse agonist FG 7142 were studied in the rat. Repeated administration of FG 7142 (15 mg/kg IP, twice daily for 10 consecutive days) induced sensitization to the effects of this drug, which from proconvulsant became a full convulsant. Thus, myoclonic seizures were observed in 30% and 80% of the animals by the third and the eighth day of treatment, respectively. The sensitization to the convulsant effect of FG 7142 persisted for up to 50 days after withdrawal and was completely prevented by the concurrent administration of the benzodiazepine receptor antagonist Ro15-1788 (15 mg/kg IP, twice a day for 10 days). Moreover, four to twelve days after withdrawal from chronic treatment with FG 7142, an increased sensitivity to the proconvulsant beta CCE and to the convulsant DMCM was observed. In addition, convulsions induced by isoniazid (350 mg/kg, SC) were potentiated in rats chronically treated with FG 7142 at 5 and 20 days after withdrawal. These pharmacological effects were paralleled by a decrease in the density of low affinity GABA receptors in the cerebral cortex and cerebellum. These results are consistent with the view that repeated administration of FG 7142 induces a long-lasting down-regulation of the GABAergic function which results in an increased sensitivity to beta-carboline inverse agonists and isoniazid. The possibility that a concomitant decrease in the responsiveness to benzodiazepines and Ro15-1788 takes place after chronic treatment with FG 7142 is also discussed.     

Blockade of the diazepam-induced increase in rat striatal acetylcholine content by the specific benzodiazepine antagonists ethyl-β-carboline-3-carboxylate and Ro 15-1788

Diazepam increased the acetylcholine content in the striatum and the hippocampus of the rat. This effect was antagonized in both brain areas by treatment with the specific central benzodiazepine blockers ethyl-β-carboline-3-carboxylate and Ro 15-1788, whereas the peripheral antagonist Ro 5-4864 was ineffective. Pretreatment with picrotoxin, a known GABA antagonist did not interface with the diazepam-induced acetylcholine increase. These results indicate a specific involvement of benzodiazepine receptors in the cholinergic action of diazepam and this effect appears to be independent of GABA receptor activation.     

Effect of a pharmacological stressor on glutamate efflux in the prefrontal cortex

The anxiogenic β-carboline, FG 7142 (20 mg/kg) significantly increased glutamate efflux in the prefrontal cortex of conscious rats as assessed by microdialysis. Pretreatment with the benzodiazepine receptor agonist, diazepam (5 mg/kg), abolished this effect. These findings indicate that anxiogenic compounds produce an effect similar to physical stressors on the outflow of glutamate, and implicate the GABA/benzodiazepine receptor complex in the stress-induced activation of glutamate systems in the prefrontal cortex.     

Changes in GABAergic transmission induced by stress, anxiogenic and anxiolytic beta-carbolines

The cerebral cortex of unstressed (handling-habituated) rats has a higher number of low affinity GABA receptors than stressed (naive) rats. Foot shock stress delivered to unstressed rats decreases the density of cortical low affinity GABA receptors to the level found in the naive animals. The effect of stress on GABA receptors is mimicked by anxiogenic beta-carbolines, both after in vitro addition (10(-6) M) to cortical membrane preparations or after the in vivo administration (20 mg/kg IP) to unstressed rats. Vice versa, benzodiazepines or anxiolytic beta-carbolines (ZK 93423, 10(-5) M) added to membranes from naive rats increase GABA binding to the level of unstressed rats and remove the decrease in the density of GABA receptors elicited by anxiogenic beta-carbolines. Rats chronically treated with the anxiogenic beta-carboline, FG 7142 (15 mg/kg IP twice a day for 10 consecutive days) have an enhanced sensitivity to punishment at 5 and 15 days after the last treatment. The behavioural effect is paralleled by a marked decrease in the total number of cortical low affinity GABA receptors. Both biochemical and behavioural effects elicited by chronic FG 7142 are prevented by the concurrent administration of the benzodiazepine antagonist Ro15-1788. These results suggest that (a) anxiolytic beta-carbolines, like benzodiazepines, increase the GABAergic transmission, (b) acute and chronic anxiogenic beta-carboline administration, like stress, decreases GABAergic transmission. Since all these effects are antagonized by the benzodiazepine receptor blocker Ro15-1788, it is tempting to speculate that stress releases an endogenous ligand for benzodiazepine recognition sites.     

Suppression of the immune response by benzodiazepine receptor inverse agonists

24 h after administration of a single dose of the benzodiazepine receptor inverse agonists N'-methyl-beta-carboline-3-carboxamide (FG 7142) and 3-carbomethoxy-4-ethyl-6,7-dimethoxy-beta-carboline (DMCM), a profound suppression of the immune response was observed in rodents. This immunosuppression was manifest as a decrease in phytohemagglutinin (PHA) and concanavalin-A (Con-A) stimulated T cell proliferation in rats and mice administered FG 7142 and a decrease in allogeneic cytotoxic T lymphocyte activity in mice administered either FG 7142 or DMCM. The effects of FG 7142 were antagonized by the prior administration of Ro 15-1788, a benzodiazepine receptor antagonist. These findings demonstrate that the neural pathways subserved by benzodiazepine receptors can modulate immune function, and suggest that these receptors may be involved in the stress-induced modulation of immune function.     

Characteristics of an atypical benzodiazepine,Ro 5-4864

Ro 5-4864 is a 1,4 benzodiazepine which, atypically, does not bind to the classical CNS benzodiazepine receptors, but has high affinity for the peripheral type of binding site found both in the periphery and in the brain. Biochemical evidence for alternative sites of action for this compound is discussed. We review the behavioral profile of Ro 5-4864 (sedative, convulsant and anxiogenic in rodents) and also describe the behavioral effects of combining Ro 5-4864 treatment with benzodiazepines (e.g., diazepam, chlordiazepoxide) and with other drugs that modify the activity of benzodiazepines (Ro 15-1788, CGS 8216, picrotoxin, PK 11195, phenytoin). In the light of these interactions and electrophysiological evidence we conclude that the actions of Ro 5-4864 are most likely to be mediated at the GABA-benzodiazepine receptor complex in the CNS.     

Reversal of increased anxiety during benzodiazepine withdrawal: evidence for an anxiogenic endogenous ligand for the benzodiazepine receptor

Rats were injected daily for 21 days with water or with chlordiazepoxide hydrochloride (10 mg/kg) and then tested in the elevated plus-maze 24-30 hr after the last of their chronic injections. At this time, rats withdrawn from chlordiazepoxide showed a significant decrease in the % of time spent on the open arms, compared with controls, thus indicating enhanced anxiety. The benzodiazepine antagonist, flumazenil (Ro 15-1788, 4 mg/kg, IP 20 min before test) significantly (p less than 0.01) reversed this withdrawal anxiety, and was without effect in the control group. The partial inverse agonist, FG 7142 (5 mg/kg IP 30 min before test) had no significant effect on the withdrawal anxiety. Possible mechanisms underlying the enhanced anxiety displayed by rats in withdrawal from chlordiazepoxide are discussed. It is concluded that a likely explanation is that chronic benzodiazepine treatment leads to an increased production and release of an endogenous ligand for the benzodiazepine receptor, with inverse agonist properties.     

Psychological stress-induced enhancement of brain lipid peroxidation via nitric oxide systems and its modulation by anxiolytic and anxiogenic drugs in mice

We investigated the effect of psychological stress on lipid peroxidation activity in the mouse brain, the mechanism underlying the psychological stress-induced change in the activity, and the effects of anxiolytic and anxiogenic drugs on the activity in psychologically-stressed animals. Psychological stress exposure using a communication box paradigm for 2-16 h significantly increased the content of thiobarbituric acid reactive substance (TBARS), an index of lipid peroxidation activity, in the brain, and the effect was maximal after peaked by a 4-h stress exposure. In the animals stressed for over 4 h, the increased brain TBARS content lasted for 30 min after the stress exposure, while no significant increase of the TBARS content was observed in the liver or serum. Trolox (67.6 mg/kg, i.p.), an antioxidant drug, but not monoamine oxidase inhibitors, clorgyline (2.5-5 mg/kg, i.p.) or 5-(4-benzylphenyl)-3-(2-cyanoethyl)-(3H)-1,3,4-oxadiazol-2-o ne (1-5 mg/kg, i.p.), significantly suppressed the effect of psychological stress. The non-selective nitric oxide (NO) synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10-100 mg/kg, i.p.) and the selective neuronal NOS inhibitor 7-nitroindazole (25 and 50 mg/kg, i.p.), but not the inducible NOS inhibitor aminoguanidine (1-100 mg/kg, i.p.), dose dependently suppressed the psychological stress-induced enhancement of lipid peroxidation in the brain. L-Arginine (300 mg/kg, i.p.), a substrate of NOS, antagonized the effect of L-NAME. Measurements of NO metabolites revealed a significant increase of NO production in the brains of stressed mice. The benzodiazepine (BZD) receptor agonist diazepam (0.05-0.5 mg/kg, i.p.), the 5-HT(1A) receptor agonists (+/-)-8-hydroxy-di-propylaminotetralin and buspirone (0.1-1 mg/kg, i. p.), but not the 5-HT(3) receptor agonist MDL72222, dose-dependently suppressed the psychological stress-induced enhancement of brain lipid peroxidation. In contrast, the administration of anxiogenic drugs, FG7142 (an inverse BZD agonist: 1-10 mg/kg, i.p.) and 1-(3-chlorophenyl)piperazine (a mixed 5-HT(2A/2B/2C) agonist: 0.1-1 mg/kg, i.p.), potentiated it. The effects of diazepam and FG7142 were abolished by the BZD receptor antagonist flumazenil (10 mg/kg, i.p.). These results indicate that psychological stress causes oxidative damage to the brain lipid via enhancing constitutive NOS-mediated production of NO, and that drugs with a BZD or 5-HT(1A) receptor agonist profile have a protective effect on oxidative brain membrane damage induced by psychological stress.     

Changes in local cerebral glucose utilization induced by the beta-carbolines FG 7142 and DMCM reveal brain structures involved in the control of anxiety and seizure activity

The brain regions that may be functionally involved in the control of anxiety and the development of seizures were examined using quantitative 1-14C-deoxyglucose autoradiography. For this purpose, beta-carbolines FG 7142 and DMCM were employed. They exert their effects via the benzodiazepine receptor, and whereas both possess anxiogenic properties, FG 7142 is a proconvulsant and DMCM a potent convulsant. The pattern of increases of local cerebral glucose utilization (LCGU) induced by FG 7142 was mainly restricted to limbic structures, such as the lateral septal nucleus, the anterior thalamic nuclei, and the mamillary nuclei. However, structures involved in motor regulation were also affected. A pronounced increase in LCGU was observed in the posterior part of the substantia nigra, pars reticulata. Further, the LCGU of the globus pallidus, the ventral thalamic nucleus, and the cerebellum was increased. DMCM likewise increased LCGU of the mamillary body and the lateral septal nucleus. In contrast to FG 7142, the hippocampal formation displayed an increase in LCGU, while LCGU of the anterior thalamic nuclei was unchanged. A pronounced increase in LCGU was seen in the substantia nigra, pars reticulata in addition to other structures functionally involved in central motor regulation. The specific benzodiazepine antagonist Ro 15-1788 antagonized the effects of both FG 7142 and DMCM. It is concluded that the beta-carbolines FG 7142 and DMCM produce selective effects upon LCGU that are mediated by benzodiazepine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of cell proliferation of human gliomas by benzodiazepines in vitro

The effects of several benzodiazepines (diazepam, clonazepam, Ro 15-1788 and Ro 5-4864) on cell proliferation of 2 human gliomas were estimated in vitro by means of [3H]-thymidine uptake assay. It was found that all tested benzodiazepines suppressed [3H]-thymidine incorporation into the DNA of glioma cells, the effects being stronger in case of peripheral-type benzodiazepine receptor ligands. The results indicated that benzodiazepines might exert an antiproliferative action on glioma tumour cells growth.     

Possible role of cholecystokinin-A receptors in regulation of thyrotropin (TSH) secretion in male rats

We studied the importance of cholecystokinin (CCK) system in the regulation of thyrotropin (TSH) and prolactin (PRL) secretion in male rats. To this end, we tested the effects of both unselective CCK agonists CCK-8 and caerulein, and CCK-B selective agonists CCK-4 and pentagastrin as well as the selective CCK antagonists (devazepide and L-365,260) at wide dose-ranges on the cold-stimulated and TRH-induced TSH and PRL secretion. Caerulein, given s.c. 15 min before sacrifice, decreased TSH levels at 5 micrograms/kg. In time course-studies, the maximum inhibition was seen at 15 min but the effect lasted at least 30, but less than 60 min. Also CCK-8 decreased TSH levels at the doses of 20 and 50 micrograms/kg at 15 min. Devazepide and L-365,260 did not affect TSH or PRL levels at any dose. The effect of caerulein (5 micrograms/kg) was antagonized by devazepide, a CCK-A antagonist, at 100 micrograms/kg, but not by a CCK-B antagonist L-365,260 tested at a wide dose range. PRL levels were not affected by any treatment. Caerulein (5 micrograms/kg), given at the same time as TRH (500 ng/kg), inhibited the TRH-induced TSH levels at 15 min, but not at 30 or 60 min. CCK-8 (50 micrograms/kg), CCK-4 (100 micrograms/kg) and pentagastrin (500 micrograms/kg) did not affect the TRH-induced TSH secretion. The results probably indicate that CCK-A receptor stimulation inhibits TSH secretion at the level of the anterior pituitary gland. PRL levels in male rats are not affected by CCK system.